1. Gain Modulation by Nicotine in Macaque V1
Anita A. Disney, Chiye Aoki, Michael J. Hawken 
Neuron 
Volume 56, Issue 4, Pages (November 2007)
DOI: /j.neuron
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1 Localization of AChRs by EM-Immunocytochemistry
(A) Labeling for the β2-nAChR subunit by silver-intensified gold (upper inset; scale bar, 10 μm) and anterogradely traced LGN terminals by ABC-DAB (middle inset; scale bar, 20 μm). (B) An EM image of a tracer-labeled terminal (t+) synapsing onto a spine (sp; note the spine apparatus, arrow), showing that tract-tracing allows identification of thalamic terminals in single planes of section in which the presynaptic element is otherwise difficult to distinguish. Silver particles (arrowheads) indicate presynaptic β2-nAChRs. Scale bar, 200 nm. (C) The quantification of β2 labeling at layer 4c synapses (x axis: sp, spine; sh, shaft; +, labeled terminals; −, unlabeled terminals). 76% of thalamic synapses onto spines in layer 4c are presynaptically immunoreactive for β2 subunits. No thalamic synapses onto dendrites are β2-nAChR-ir. Layer 4c thalamic synapses are immunonegative for m1 (D) and m2 (E) muscarinic receptors.
Neuron  , DOI: ( /j.neuron )
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3. Figure 2 Expression of Nicotinic Receptors by V1 Interneurons
(A) Dual immunofluorescence from layer 3. The red channel (left) shows GABA immunoreactivity; the green channel (middle) shows β2-nAChR-immunoreactivity. In the merged image (right), dual-labeling appears yellow. In every layer the proportion of β2-nAChR-ir somata that are also GABA-ir exceeds 60% (B). By EM there is prominent β2-nAChR immunolabeling of putatively inhibitory dendrites—in (C), a layer 4cβ shaft (d) receives direct asymmetric synapses (black asterisks), suggesting that it is the dendrite of a GABAergic (aspiny) neuron. The dendrite is densely β2-nAChR immunoreactive (black arrowheads). Also visible is an asymmetric synapse onto a spine (sp) that has presynaptic β2-nAChR immunoreactivity in the terminal (t) cytoplasm. The empty space inside the terminal (s) is the tissue surface—data were collected very close to the tissue/EPON interface. Scale bar, 200 nm. (D) Some β2-nAChR-ir neurons express the calcium-binding proteins (CBP) calbindin (CB) (left; 111 of 641 neurons, 17%) and parvalbumin (PV) (right; 76 of 254 neurons, 30%). In contrast, very few β2-nAChR-ir neurons in any layer express calretinin (CR) (middle; 25 of 453 neurons, 6%). All error bars = SEM.
Neuron  , DOI: ( /j.neuron )
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4. Figure 3 The Effect of Nicotine on Visual Gain in V1
(A) The average, across neurons, of the summed contrast responses (area under the contrast-response curve) obtained at the maximum applied iontophoretic current. Data are normalized to the response obtained before any nicotine was applied. Nicotine produces an increase in gain in layer 4c but not in any other layer (number of sites in parentheses above each bar).
(B) The development of the nicotine effect in 4c across repeats of contrast-response measurement.
(C) Normalized contrast-response area was stationary in the absence of nicotine. Only data for sites enhanced by nicotine are shown in (C) (i.e., not all 4c sites as in [A] and [B]; see main text).
y axis, all graphs: normalized area. All error bars = SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2007 Elsevier Inc. Terms and Conditions

5. Figure 4 Nicotine Effects Were Dose Responsive and Recovered Rapidly
(A) The averaged, normalized response area as a function of ejection current for enhanced, suppressed, and unaffected neurons.
(B) Nicotine iontophoresis was always followed by recovery runs in which the contrast-response function was measured without iontophoresis of any substance. These data come from the first recovery run after nicotine was delivered. At enhanced sites, high ejection currents often produced a recovery overshoot (below baseline), but enhancement was still seen in subsequent nicotine runs. (Note: first open square is labeled “recovery” because the order of delivery of nicotine doses varied from neuron to neuron).
All error bars = SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2007 Elsevier Inc. Terms and Conditions

6. Figure 5 The Gain Enhancement in Layer 4c Is Not a pH Effect
Combined current and pH controls were run for 38 (of 72) neurons, including 12 (of 22) enhanced sites. A saline solution, adjusted to pH 3.0, was ejected using the same range of currents as was used to eject nicotine. In this way the effects of pH and current are both examined. (A) For the 12 enhanced sites, the pH 3.0 saline was weakly excitatory (diamonds), but the magnitude of the effect was 10-fold smaller than that observed for nicotine (squares). The peak normalized response areas (both obtained at 80 nA) were as follows: pH 3.0 saline = 1.096, nicotine = At very high ejection currents (>80 nA), the pH 3.0 solution was actually slightly suppressive. n = 8 for the 120–159 nA data point. In (B), it can be seen that the effect of the pH 3.0 solution on normalized response area did not differ between enhanced (squares) and unaffected (diamonds) sites. In addition, in preliminary experiments using the broad cholinergic agonists ACh (gray triangles; pH 5.0) and carbachol (circles; pH 6.5), we observed a similar enhancement in layer 4c with nonacidic solutions (C). Our preliminary antagonist studies also show that the effect of nicotine is eliminated following receptor blockade. (D) Data for an example cell in which mecamylamine was used as an nAChR antagonist. Fits were obtained for contrast-response functions under baseline conditions (filled gray circles/solid lines) and during iontophoresis of nicotine (black circles/lines; 40 nA) or nicotine (40 nA) plus mecamylamine (120 nA; open gray circles/dashed lines). The effect of nicotine was completely blocked by mecamylamine in this cell, as it was for five other cells ([E]; effect was blocked in six of eight enhanced cells; data shown is the average across all eight cells tested with mecamylamine).
Error bars = SEM for (A)–(C) and (E), SD in (D).
Neuron  , DOI: ( /j.neuron )
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7. Figure 6 Parametric Analysis: Single-Cell Example
Spike responses were averaged and fit with a Naka-Rushton function (see Experimental Procedures). The parameters Rmax, c50, and n are the maximum firing rate and the contrast and slope at half-maximum, respectively; the parameter sFR captures offset attributable to background activity. (A) The fits obtained for a representative cell from layer 4c. The nicotine curve (black) is shifted to the left—indicating increased sensitivity—and shows a higher maximum response than baseline (gray). Error bars = SD. A bootstrap analysis (1000 samples) performed on data for this cell (see Experimental Procedures) shows that nicotine alters Rmax (B), but not c50 (C).
Neuron  , DOI: ( /j.neuron )
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8. Figure 7 Population Data on Rmax Changes from Parametric Analysis
(A) A bootstrap analysis on the Rmax parameter of fits from the baseline (x axis) and nicotine (y axis) conditions shows that Rmax is elevated by nicotine at most layer 4c sites (black circles). In contrast, the data from other layers (gray circles) is clustered around the unity line. Nicotine did not alter c50 in any layer (B). Data from 15 recording sites for which histological reconstruction was not possible is not shown. None of the omitted sites showed a significant change in Rmax or c50.
Neuron  , DOI: ( /j.neuron )
Copyright © 2007 Elsevier Inc. Terms and Conditions

9. Figure 8 Nicotine Lowers Detection Thresholds for Neurons in Layer 4c
Fits are shown for three cells in layer 4c (A–C) and for the population of cells that showed Rmax changes (D) with nicotine data in black and baseline data in gray. Spontaneous firing rates in (A)–(C) are indicated by the filled squares at the far left of each graph (“Sp” on the x axis) and by the dashed lines extending from the squares. For all three cells, the contrast at which the nicotine response reliably rises above the threshold set by the background activity (black arrows, set to the first point for which the error bars clear the dashed line) is lower than the corresponding threshold contrast under baseline conditions. Error bars = SD. In (D), data from the neurons that showed changes in Rmax (p < 0.1) have been normalized to the maximum firing rate obtained under baseline conditions, averaged, and fit. Spontaneous activity was not significantly altered in these neurons and the normalized spontaneous rate has thus been averaged across both conditions—the value shown is this mean plus 3 SD (dashed line). The threshold contrast defined by this level of background activity is lower (∼2% contrast) with nicotine than under baseline conditions (∼4%). Closer inspection reveals that this is the case regardless of the chosen value for threshold contrast.
Neuron  , DOI: ( /j.neuron )
Copyright © 2007 Elsevier Inc. Terms and Conditions